Antibody for detecting epithelial ovarian cancer marker and method for diagnosing epithelial ovarian cancer

ABSTRACT

It is intended to find a highly specific epithelial ovarian cancer marker and to provide an antibody capable of specifically recognizing and detecting the marker or a fragment of the antibody. The present invention provides an anti-β1,6-N-acetylglucosaminyltransferase 5B antibody for diagnosis of epithelial ovarian cancer, i.e., an antibody for detection of a glycosyltransferase β1,6-N-acetylglucosaminyltransferase 5B as an epithelial ovarian cancer marker. The antibody recognizes, as an epitope, a part of a polypeptide of the enzyme consisting of the amino acid sequence represented by SEQ ID NO: 1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. § 371of PCT/JP2012/068990, filed Jul. 26, 2012, which claims benefit ofJapanese Appl. No. 2011-163323, filed Jul. 26, 2011, which isincorporated herein by reference.

SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby is incorporated by referenceinto the specification. The name of the text file containing theSequence Listing is 14234871_Repl_Seq_List_ST25.txt. The size of thetext file is 17 KB, and the text file was created on Mar. 10, 2014.

TECHNICAL FIELD

The present invention relates to ananti-β1,6-N-acetylglucosaminyltransferase 5B antibody for detection ofan epithelial ovarian cancer marker, a hybridoma producing the antibody,and a method for diagnosis of epithelial ovarian cancer using theantibody.

BACKGROUND ART

Ovarian cancer is a cancer with a low incidence, compared with breastcancer or uterine cancer, among gynecologic cancers. Both incidence andmortality of this cancer, however, have been on the increase in recentyears. In general, ovarian cancer is substantially asymptomatic early inthe course of the disease and often found at the already advanced stageof symptoms. Hence, this disease has poor prognosis and exhibits thehighest mortality among gynecologic cancers. Ovarian cancer is known toinclude, for example, surface epithelial-stromal tumors (hereinafter,referred to as “epithelial ovarian cancer”) developed from surfaceepithelial cells in the ovary and germ cell tumors developed from germcells, depending on the area affected. Of them, epithelial ovariancancer accounts for approximately 90% of all ovarian cancer cases and isoften seen particularly in middle-aged women in their 40s or older.Thus, the early detection of epithelial ovarian cancer is important forthe treatment of the disease.

The ovary is an organ that has no contact with the outside of the body.Unlike the uterus, the ovary can be neither examined endoscopically norsubjected to cell harvest without abdominal section or perforation. Inaddition, epithelial ovarian cancer is generally difficult to detect bypalpation before the ovary is enlarged at the advanced stage ofsymptoms. Hence, epithelial ovarian cancer is often undetected inordinary examination or diagnosis methods. Although echography, MRI, CT,or the like is relatively effective for the early detection ofepithelial ovarian cancer, this examination itself is extensive workwith a high cost. In addition, these approaches, unfortunately, do notalways have high diagnostic accuracy to distinguish between benign andmalignant tumors.

Against this backdrop, tumor markers have received attention in recentyears. The tumor markers refer to substances that are produced by cancercells or produced by cells surrounding cancer cells in response to thecancer cells. The abundance of each tumor marker in a body fluid canreflect the presence or absence of tumor or the prognosis thereof andtherefore serve as an index for, for example, cancer diagnosis anddecision on therapeutic strategies. Also, this approach permitsexamination using a body fluid and is thus relatively low invasive.Advantageously, this examination is also convenient and low in cost.

Tumor markers composed of proteins such as CA125, CA602, CA130, CA72-4,CA546, CA 19-9, and STN have been known so far as tumor markers forepithelial ovarian cancer (Non Patent Literatures 1 to 6). Methods fordiagnosis of cancer using these tumor markers typically involvemeasuring the expression levels of the tumor markers in the serum ofnormal individuals and epithelial ovarian cancer patients anddetermining the presence or absence of cancer developed in a testsubject on the basis of the difference in the levels.

These proteins, however, present specificity problems in such a way thatCA125 exhibits positivity to a non-cancer benign gynecologic diseasesuch as endometriosis or CA72-4, CA19-9, and STN exhibit positivity tovarious cancers of the digestive system including the stomach and thelarge intestine in addition to ovarian cancer. Epithelial ovarian canceris further classified into serous, clear cell, mucinous, andendometrioid tumors depending on the histological type. The markersdiffer in reactivity among these histological types and therefore, donot correctly reflect the progression of cancer in some cases. Forexample, the ovarian cancer markers such as CA125, CA602, and CA546 havea low positive rate for mucinous ovarian cancer. Unfortunately, thishistological type is therefore rarely detected even at the advancedstage.

Patent Literature 1 discloses a monoclonal antibody for use in thediagnosis of cancers including ovarian cancer against humangalactosyltransferase associated with tumor (GAT) as a tumor marker, ahybridoma producing the antibody, and a method for assaying humangalactosyltransferase associated with tumor in a specimen using theantibody. Also, Patent Literature 2 discloses a method for detectinggynecologic cancers early using glycosyltransferasesβ1,3-galactosyltransferase 5, β1,3-galactosyltransferase 4, andN-acetylglucosamine-6-O-sulfotransferase 2 as tumor markers. Theseglycosyltransferases used as tumor markers in Patent Literatures 1 and 2are enzymes that synthesize sugar chains to be bound with, for example,glycoproteins, glycolipids or proteoglycans, and are usually anchored onGolgi membranes as membrane proteins localized to the Golgi bodies.Thus, these enzymes are not secreted to the outside of the cells and aretherefore rarely detected in the body fluids of normal individuals. Inovarian cancer, however, it is known that glycosyltransferases areabnormally cleaved due to the increased expression level of a certainkind of protease and released to the outside of the cells. As a result,significant amounts of glycosyltransferase fragments are detected inbody fluids.

The glycosyltransferase β1,4-galactosyltransferase described in PatentLiterature 1 has an exceedingly high expression level amongglycosyltransferases and is thus secreted in large amounts to theoutside of the cell. Hence, this glycosyltransferase exhibits a serumconcentration of approximately 200 ng/mL even in normal individuals andthus fails to distinguish between benign disease and cancer by itsenzymatic activity alone. Accordingly, Patent Literature 1 was focusedon the presence of a fragment of the abnormally cleavedglycosyltransferase in the culture supernatant of ovarian cancer cellsand the ascitic fluid of an ovarian cancer patient. An antibody thatrecognizes only this fragment was used to attempt the construction of anassay system highly specific for ovarian cancer. Nonetheless, acommercially available clinical diagnosis kit based on this assay systemexhibited positivity even for healthy women in some cases (Non PatentLiterature 7). For this reason, GAT is currently used mainly in themonitoring of ovarian cancer recurrence and rarely used as a marker forearly detection.

In Patent Literature 2, an attempt was made to detect marker candidateproteins in the blood of gynecologic cancer patients on the basis ofreports stating that the expression of various proteins includingglycosyltransferases is generally increased or decreased in cancertissues. As a result, two glycosyltransferases were found to be usefulin assay. The literature discloses an assay method according to thefindings. In this case, however, the expression or synthesis products ofthese glycosyltransferases are reportedly related to digestive systemcancers rather than gynecologic cancers. Although their expression wasthen confirmed in ovarian cancer cell lines (Non Patent Literature 8),there has been no report on the comparison of the expression levelsbetween ovarian cancer or other gynecologic cancers and digestive systemcancers.

As mentioned above, Patent Literatures 1 and 2 are directed only to thedetection of markers in the body fluids of ovarian cancer patients withlittle consideration given to glycosyltransferase expression in ovariancancer and disclose a glycosyltransferase fragment that happened to bemeasurable, as an ovarian cancer marker. This may lead to specificityproblems as ovarian cancer markers.

CITATION LIST Patent Literature

-   Patent Literature 1: JP Patent Publication (Kokai) No. 3-259093 A    (1991)-   Patent Literature 2: WO2009/028417

Non Patent Literature

-   Non Patent Literature 1: Bast R. C. Jr. et al., 1983, N. Engl. J.    Med., 309: 883-887-   Non Patent Literature 2: Suzuki M. et al., 1990, Nippon Gan Chiryo    Gakkai Shi, 25: 1454-1460-   Non Patent Literature 3: Inaba N. et al., 1989, Nippon Gan Chiryo    Gakkai Shi, 24: 2426-2435-   Non Patent Literature 4: Ohuchi N. et al., 1988, Gan To Kagaku    Ryoho, 15, 2767-2772-   Non Patent Literature 5: Nozawa S. et al., 1996, Nippon Rinsho, 54:    1665-1673-   Non Patent Literature 6: Charpin C. et al., 1982, Int. J. Gynecol.    Pathol., 1: 231-245-   Non Patent Literature 7: Konica Minolta, Inc., GAT Test Kit product    document-   Non Patent Literature 8: Seko A. et al., 2009, Tumor Biol., 30:    43-50

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide an antibody or afragment of the antibody capable of specifically recognizing andquantitatively and/or qualitatively detecting a tumor marker highlyspecific for epithelial ovarian cancer.

Another object of the present invention is to provide a method forconveniently and relatively low invasively diagnosis with high accuracyrate whether or not a test subject has epithelial ovarian cancer byquantitatively and/or qualitatively detecting an epithelial ovariancancer marker using the antibody or the fragment thereof.

Solution to Problem

The present inventors have conducted diligent studies and consequentlyfound that a glycosyltransferase β1,6-N-acetylglucosaminyltransferase 5B(hereinafter, the enzyme is also abbreviated to “MGAT5B” in the presentspecification) is present in larger amounts in the samples of epithelialovarian cancer patients compared with normal individuals. The presentinvention is based on the findings and provides the following:

(1) An antibody for detection of an epithelial ovarian cancer markerrecognizing, as an epitope, a part of a polypeptide consisting of theamino acid sequence represented by SEQ ID NO: 1.

(2) The antibody according to (1), wherein the antibody is a monoclonalantibody.

(3) The antibody according to (2), wherein the antibody is produced by ahybridoma identified by International Accession No. FERM ABP-11496, FERMABP-11497, FERM ABP-11498, or FERM ABP-11499.

(4) A recombinant anti-MGAT5B antibody for detection of an epithelialovarian cancer marker comprising at least one set of corresponding lightchain complementarity determining regions and heavy chaincomplementarity determining regions in an antibody according to any of(1) to (3).

(5) An antibody fragment for detection of an epithelial ovarian cancermarker, which is a fragment of an antibody according to any of (1) to(3) or a recombinant antibody according to (4) and having the activityof specifically recognizing MGAT5B.

(6) A hybridoma producing an antibody according to (2).

(7) The hybridoma according to (6), wherein the hybridoma is identifiedby International Accession No. FERM ABP-11496.

(8) The hybridoma according to (6), wherein the hybridoma is identifiedby International Accession No. FERM ABP-11497.

(9) The hybridoma according to (6), wherein the hybridoma is identifiedby International Accession No. FERM ABP-11498.

(10) The hybridoma according to (6), wherein the hybridoma is identifiedby International Accession No. FERM ABP-11499.

(11) A method for diagnosis of epithelial ovarian cancer, comprisingquantitatively and/or qualitatively detecting a MGAT5B polypeptidefragment present in a sample derived from a test subject, anddetermining the presence or absence of epithelial ovarian cancerdeveloped in the test subject on the basis of the detection results.

(12) The method for diagnosis of epithelial ovarian cancer according to(11), wherein when the quantitative detection results of the MGAT5Bpolypeptide fragment show a quantification value of the MGAT5Bpolypeptide fragment equal to or higher than a predetermined value, thetest subject is determined to be likely to have epithelial ovariancancer.

(13) The method for diagnosis of epithelial ovarian cancer according to(11) or (12), wherein the MGAT5B polypeptide fragment is the whole or apart of a polypeptide consisting of the amino acid sequence representedby SEQ ID NO: 1.

(14) The method for diagnosis of epithelial ovarian cancer according toany of (11) to (13), wherein the MGAT5B polypeptide fragment is detectedusing at least one antibody, recombinant antibody, and/or antibodyfragment selected from the group consisting of an antibody according toany of (1) to (3), a recombinant antibody according to (4), and anantibody fragment according to (5).

(15) The method for diagnosis of epithelial ovarian cancer according to(14), wherein two antibodies, recombinant antibodies, and/or antibodyfragments that recognize different epitopes on the MGAT5B polypeptidefragment are used.

(16) The method for diagnosis of epithelial ovarian cancer according toany of (11) to (15), wherein the sample is a body fluid, a peritoneallavage fluid, or a tissue.

The present specification encompasses the contents described in thespecification and/or drawings of Japanese Patent Application No.2011-163323 on which the priority of the present application is based.

Effects of Invention

The antibody of the present invention and/or the fragment thereof iscapable of specifically recognizing and detecting a glycosyltransferaseMGAT5B polypeptide fragment as an epithelial ovarian cancer marker.

The hybridoma of the present invention can stably supply an anti-MGAT5Bmonoclonal antibody capable of specifically recognizing and detecting aglycosyltransferase MGAT5B as an epithelial ovarian cancer marker.

The method for diagnosis of epithelial ovarian cancer according to thepresent invention can detect epithelial ovarian cancer conveniently andrelatively low invasively with high accuracy rate. As a result, whetheror not a test subject has epithelial ovarian cancer can be determinedearly.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of Western blotting by which anti-MGAT5B monoclonalantibodies (a: GT131-12 antibody and b: GT131-18 antibody) derived fromhybridomas obtained in Example 1 were studied for their antigenspecificity. Lane 1 shows the results about a FLAG-MGAT5B polypeptidefragment. Lane 2 shows the results about a MGAT5B polypeptide fragmentfrom which the FLAG tag was cleaved off by enzymatic treatment. Lane 3shows the results about the immunoglobulin- and albumin-free normalhuman serum (NHS). Lane 4 shows the results about the untreated NHS.

FIG. 2 shows results of Western blotting by which FLAG-MGAT5Bpolypeptide fragments immunoprecipitated with anti-MGAT5B antibodieswere detected using various antibodies (a: anti-FLAG antibody, b:GT131-12 antibody, and c: GT131-18 antibody). Lane 1 shows the resultsabout a control FLAG-MGAT5B polypeptide fragment. Lane 2 shows theresults about a sample obtained by the immunoprecipitation with aGT131-2 antibody of a FLAG-MGAT5B polypeptide fragment mixed with theserum of a normal individual. Lane 3 shows the results about a sampleobtained by the immunoprecipitation with a GT131-7 antibody of aFLAG-MGAT5B polypeptide fragment mixed with the NHS. Lane 4 shows theresults about a sample obtained by the immunoprecipitation with aGT131-12 antibody of a FLAG-MGAT5B polypeptide fragment mixed with theNHS. Lane 5 shows the results about a sample obtained by theimmunoprecipitation with a GT131-18 antibody of a FLAG-GAT5B polypeptidefragment mixed with the NHS. Lane 6 shows the results about anon-immunoprecipitated FLAG-MGAT5B polypeptide fragment mixed with theNHS.

FIG. 3 shows the immunostaining of an ovarian cancer cell line usinganti-MGAT5B antibodies. FIG. 3a is a staining pattern of an epithelialovarian cancer cell line RMUG-S immunostained with a GT131-2 antibody.FIG. 3b is a staining pattern of the cell line immunostained with aGT131-18 antibody. FIG. 3c is a staining pattern of the cell lineimmunostained with a positive control MAb8628 antibody.

FIG. 4 shows signal intensity vs. a concentration standard in study onthe combination of antibodies for sandwich ELISA.

FIG. 5 shows a calibration curve of MGAT5B polypeptide fragmentsobtained from values of a concentration standard measured by sandwichCLEIA using a GT131-7 antibody and a GT131-12 antibody.

FIG. 6 shows the concentration of the epithelial ovarian cancer markerMGAT5B polypeptide fragment of the present invention in a peritoneallavage fluid collected from a test subject, wherein the concentrationwas measured by sandwich CLEIA using a GT131-7 antibody and a GT131-12antibody.

FIG. 7 shows the correlation between a measurement value of the MGAT5Bpolypeptide fragment and a measurement value of an existing ovariancancer marker CA125 (a) or GAT (b) in peritoneal lavage fluids collectedfrom epithelial ovarian cancer patients.

FIG. 8 shows the correlation between a CA125 measurement value and a GATmeasurement value in peritoneal lavage fluids collected from epithelialovarian cancer patients.

DESCRIPTION OF EMBODIMENTS

1. Antibody for Detection of Epithelial Ovarian Cancer Marker

1-1. Definition and Constitution

The first embodiment of the present invention relates to an antibody fordetection of an epithelial ovarian cancer marker. The antibody of thepresent invention is an antibody that is used for detecting anepithelial ovarian cancer marker and recognizes and specifically bindsto an epitope contained in the epithelial ovarian cancer marker.

In the present invention, the “epithelial ovarian cancer marker” refersto a biological marker for detection of epithelial ovarian cancer whichis a biomaterial serving as an index showing that a test subject hasepithelial ovarian cancer. The epithelial ovarian cancer markeraccording to the present invention is specifically aβ1,6-N-acetylglucosaminyltransferase 5B polypeptide fragment.

“β1,6-N-acetylglucosaminyltransferase 5B (MGAT5B)” is one type ofN-linked sugar chain-synthesizing enzyme, and as a membrane proteinmainly localized to the Golgi membrane. This enzyme transfersN-acetylglucosamine with the β1-6 linkage (Kaneko M. et al., 2003, FEBSLetters, 554: 515-519). MGAT5B contains one N-terminal transmembranedomain and has an enzymatically active region as a C-terminal region.The MGAT5B according to the present invention is the human-derivedwild-type MGAT5B composed of 790 amino acids and registered underGenBank Accession No. NP_653278. Specifically, the MGAT5B according tothe present invention is a polypeptide consisting of the amino acidsequence represented by SEQ ID NO: 2. In the present specification,MGAT5B encompasses wild-type MGAT5B and natural variants thereof.

In the present specification, the “natural variant” of MGAT5B refers toa naturally occurring variant that contains the deletion, substitution,addition, or insertion of 1 to 10, preferably 1 to 5, more preferably 1to 4, 1 to 3, or 1 or 2 amino acids in the amino acid sequence (SEQ IDNO: 2) constituting wild-type MGAT5B or is a polypeptide exhibitingapproximately 90% or higher, preferably approximately 95% or higher,more preferably approximately 98% or higher identity to the amino acidsequence represented by SEQ ID NO: 2. In this context, the “identity”refers to the ratio (%) of identical amino acid residues of a targetamino acid sequence to the total number of amino acid residues of theamino acid sequence represented by SEQ ID NO: 2 including the number ofgaps when the amino acid sequence represented by SEQ ID NO: 2 and thetarget amino acid sequence are aligned such that the maximum degree ofidentity can be achieved with or without introduced gaps. This identitycan be determined using a protein search system based on BLASTP orFASTA. Specific examples of the natural variant include variants basedon polymorphisms such as SNP (single-nucleotide polymorphism) andsplicing variants. In this context, the natural variant of MGAT5B is notnecessarily required to have enzymatic activity equivalent to that ofwild-type MGAT5B. This is because, reportedly, some natural variants ofglycosyltransferases substantially lose activity even due to thesubstitution of only one amino acid (Nishihara S. et al., 1993, Biochem.Biophys. Res. Commun. 196: 624-631).

In the present specification, the “MGAT5B polypeptide fragment” refersto a MGAT5B-derived polypeptide fragment that is released to the outsideof the epithelial ovarian cancer cell as a result of abnormal cleavageby protease from the Golgi membrane in the cell and is capable offunctioning as an epithelial ovarian cancer marker as mentioned above.Specifically, the MGAT5B polypeptide fragment and a fragment derivedfrom the polypeptide correspond to a polypeptide consisting of the wholeor a part of the intra-Golgi C-terminal region of MGAT5B except for theN-terminal region containing the transmembrane domain, for example, apolypeptide consisting of the whole or a part of an amino acid sequence(SEQ ID NO: 1) from glycine at residue 51 (counted with initiatingmethionine as the 1st position) to downstream residues in human MGAT5Bshown in SEQ ID NO: 2.

The “antibody for detection of epithelial ovarian cancer” of the presentinvention refers to an antibody that is induced with the epithelialovarian cancer marker MGAT5B polypeptide fragment as an antigen. Thus,in the present specification, the “antibody for detection of anepithelial ovarian cancer marker” is also referred to as an “anti-MGAT5Bantibody”. The anti-MGAT5B antibody is capable of recognizing andbinding to a part of the epithelial ovarian cancer marker as an epitopeand specifically detecting the epithelial ovarian cancer marker.Specifically, a part of an amino acid consisting of the amino acidsequence represented by SEQ ID NO: 1 is used as an epitope. In thiscontext, the term “a part” refers to one or more regions each composedof 5 to 15, preferably 5 to 10, more preferably 6 to 10 consecutiveamino acids.

The anti-MGAT5B antibody of the present invention may be any ofpolyclonal and monoclonal antibodies. A monoclonal antibody is desirablefor achieving more specific detection. Specific examples of theanti-MGAT5B monoclonal antibody include anti-MGAT5B antibodies producedby hybridomas identified by International Accession No. FERM ABP-11496,FERM ABP-11497, FERM ABP-11498, and FERM ABP-11499. These antibodieswill be described in detail in the paragraph “(3) Preparation ofanti-MGAT5B monoclonal antibody”.

The anti-MGAT5B antibody of the present invention can be modified. Inthis context, the “modification” includes functional modificationnecessary for antigen-specific binding activation, such asglycosylation, and labeling necessary for antibody detection. Examplesof the antibody labeling include labeling using fluorescent dyes (FITC,rhodamine, Texas Red, Cy3, and Cy5), fluorescent proteins (e.g., PE,APC, and GFP), enzymes (e.g., horseradish peroxidase, alkalinephosphatase, and glucose oxidase), and biotin or (strept)avidin.

The modification on the anti-MGAT5B antibody may be altered. Forexample, the glycosylation of the anti-MGAT5B antibody may be altered inorder to adjust the affinity of the anti-MGAT5B antibody for the targetantigen epithelial ovarian cancer marker. Specifically, examples thereofinclude alteration by which the glycosylation site in each frameworkregion (FR) of the anti-MGAT5B antibody is removed by the introductionof substitution into amino acid residue(s) constituting theglycosylation site to thereby delete glycosylation at the site.

Preferably, the anti-MGAT5B antibody of the present invention hasaffinity for the epithelial ovarian cancer marker as high as adissociation constant of 10⁻⁸ M or lower, preferably 10⁻⁹ M or lower,more preferably 10⁻¹⁰ M or lower. The dissociation constant can bemeasured using a technique known in the art. The dissociation constantmay be measured using, for example, rate evaluation kit software ofBIAcore system (GE Healthcare Japan Corp.).

The anti-MGAT5B polyclonal antibody or the anti-MGAT5B monoclonalantibody of the present invention can be obtained by a production methodmentioned later. Alternatively, the anti-MGAT5B monoclonal antibody maybe prepared by a chemical synthesis method or a recombinant DNAtechnique on the basis of its amino acid sequence. The anti-MGAT5Bmonoclonal antibody may also be obtained from a hybridoma producing theantibody.

The anti-MGAT5B antibody of the present invention is not particularlylimited by an organism species of origin. The anti-MGAT5B antibody ofthe present invention can be derived from every animal source includingbird and mammals. Examples of such animal sources include mice, rats,guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens,and humans. The anti-MGAT5B antibody of the present invention is notparticularly limited by a globulin type and can be any of IgG, IgM, IgA,IgE, IgD, and IgY. IgG and IgM are preferred.

1-2. Preparation of Anti-MGAT5B Antibody

The anti-MGAT5B antibody of the present invention, i.e., the anti-MGAT5Bpolyclonal antibody or the anti-MGAT5B monoclonal antibody, or thehybridoma producing the anti-MGAT5B monoclonal antibody can be preparedby a method described below. However, the production method of thepresent invention is not limited to the method described below, and anyother method known in the art can be used in the preparation of theantibody or the hybridoma.

(1) Preparation of Immunogen

The epithelial ovarian cancer marker is prepared as an immunogen. In thepresent invention, the epithelial ovarian cancer marker that may be usedas an immunogen is, for example, the whole or a part of a human MGAT5Bpolypeptide fragment having the amino acid sequence represented by SEQID NO: 1, or the whole or a part of a variant polypeptide fragmentthereof.

The MGAT5B polypeptide fragment as an immunogen can be prepared using,for example, a chemical synthesis method or a DNA recombinationtechnique.

In the case of preparing the fragment using the chemical synthesismethod, an appropriate MGAT5B polypeptide fragment for use as animmunogen can be chemically synthesized by an approach known in the art,for example, a solid-phase peptide synthesis method, on the basis ofinformation about, for example, the amino acid sequence of SEQ ID NO: 1.

In the case of preparing the fragment using the DNA recombinationtechnique, a MGAT5B-encoding cDNA (MGAT5B cDNA) can be incorporated intoan appropriate expression system and expressed to obtain the MGAT5Bpolypeptide fragment. Hereinafter, the method for preparing the MGAT5Bpolypeptide fragment will be described with reference to specificexamples.

(Preparation of MGAT5B cDNA)

The MGAT5B cDNA can be prepared by a technique known in the art, forexample, a cDNA cloning method. Specifically, a human cDNA library isfirst prepared. The human cDNA library can be prepared by: extractingtotal RNAs according to a routine method from, for example, humanfibroblasts (neuroblastoma cell line SK-N-SH) expressing the MGAT5B geneor the like; then recovering poly-A(+) RNAs by treatment with oligo dTcellulose columns; and performing RT-PCR with the recovered RNAs astemplates. Alternatively, a commercially available human cDNA librarymay be used.

Subsequently, the MGAT5B cDNA clone of interest is isolated from thehuman cDNA library. Specifically, the clone can be isolated by ascreening method known in the art, for example, a hybridizationscreening method, an expression screening method, or an antibodyscreening method, using primers and/or a probe appropriately designed onthe basis of the MGAT5B gene sequence. The MGAT5B gene sequence isregistered under Accession No. NM_144677 in the GenBank database. Theprimers are designed so that a MGAT5B gene region encoding the aminoacid sequence represented by SEQ ID NO: 1 is incorporated in anamplification fragment. An appropriate restriction site for cloningafter isolation or a tag sequence for protein purification (FLAG, HA,His, myc, GFP, etc.) may be introduced to the 5′ end of the forward orreverse primer. Also, the probe is designed with care so that a nucleicacid sequence encoding the amino acid sequence represented by SEQ ID NO:1 in the MGAT5B gene is incorporated in an amplification region. Theisolated MGAT5B cDNA clone may be amplified, if necessary, by a nucleicacid amplification method such as PCR.

The details of the cDNA cloning technique are described in, for example,Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory ManualSecond Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. The description thereof can therefore be referred to.

(Preparation of MGAT5B Expression Vector)

Next, the MGAT5B cDNA clone thus obtained is incorporated into anexpression vector. Specifically, examples thereof include plasmid andviral expression vectors. An expression vector for E. coli (e.g., pET21αseries, pGEX4T series, pUC118 series, pUC119 series, pUC18 series, andpUC19 series), a Bacillus subtilis-derived expression vector (e.g.,pUB110 series and pTP5 series), a yeast-derived expression vector (e.g.,YEp13 series, YEp24 series, and YCp50 series), an expression vector forinsect cells (e.g., baculovirus), or an expression vector for animalcells (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo) can be usedas the expression vector according to an expression host. The expressionvector can usually contain, for example, a promoter, a terminator, anenhancer, a polyadenylation signal, a replication origin, and aselection marker, as regulatory elements. Also, the expression vectorused may have a multicloning site for cloning of the cDNA fragment ofinterest or a tag sequence at the 5′ or 3′ end of the cDNA fragmentinsertion site for expression as a fusion polypeptide with a labelingpeptide (tag) that facilitates purification. The expression vector usedmay further have a sequence encoding a secretory signal sequence, at the5′ end of the insertion site. As a result, an expressed maturepolypeptide can be extracellularly secreted. Such expression vectors orother expression systems are commercially available as useful productsfrom each manufacturer (Takara Bio Inc., Daiichi Pure Chemicals Co.,Ltd., Agilent Technologies, Inc., Merck KGaA, Qiagen N.V., Promega K.K.,Roche Diagnostics K.K., Life Technologies Corp., GE Healthcare JapanCorp., etc.). These products may therefore be used. For the insertion ofthe MGAT5B cDNA to the expression vector, the purified MGAT5B cDNA canbe cleaved with appropriate restriction enzymes and inserted to acorresponding appropriate restriction site in the expression vector toligate the cDNA fragment with the vector. If necessary, the MGAT5B cDNAmay be subcloned using an appropriate plasmid or the like before theincorporation into the expression vector.

The details of the cDNA cloning technique are also described in, forexample, Sambrook, J. et al., (1989) Molecular Cloning: A LaboratoryManual Second Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. The description thereof can therefore be referred to.

(Expression of MGAT5B Polypeptide Fragment in Host Cell)

Subsequently, the obtained MGAT5B expression system (e.g., MGAT5Bexpression vector) is transferred to host cells to express the MGAT5Bpolypeptide fragment of interest serving as an immunogen.

The host cells used are not particularly limited as long as the hostcells are adaptable to the expression vector used in the preparation ofthe MGAT5B expression vector and can express MGAT5B. For example,Escherichia coli (E. coli) can be used for the expression vector for E.coli used. Bacillus subtilis can be used for the Bacillussubtilis-derived expression vector used. Yeast (e.g., budding yeast:Saccharomyces cerevisiae and fission yeast: Schizosaccharomyces pombe)can be used for the yeast-derived expression vector used. Insect cells(e.g., Sf cells) can be used for the expression vector for insect cellsused. Mammalian cells (e.g., HEK293, HeLa, COS, CHO, and BHK) or thelike can be used for the expression vector for animal cells used.Alternatively, a cell-free translation system may be used. The MGAT5Bexpression vector can be transferred to the host cells according to aDNA transfer method known in the art for each host cell withoutparticular limitation. Examples of the method for transferring thevector to bacteria include a heat shock method, a calcium ion method,and electroporation. All of these techniques are known in the art anddescribed in various literatures including Sambrook, J. et al., (1989)Molecular Cloning: A Laboratory Manual Second Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. For example, a Lipofectinmethod (PNAS (1989) Vol. 86, 6077), electroporation, a calcium phosphatemethod (Virology (1973) Vol. 52, 456-467), or DEAE-dextran method ispreferably used as a method for transferring the MGAT5B expressionvector to animal cells. Alternatively, a commercially available nucleicacid transfer agent such as Lipofectamine 2000 (Life Technologies Corp.)may be used. Transformants for MGAT5B expression can be obtained bythese procedures.

When the obtained transformants for MGAT5B expression are microbes suchas E. coli or yeast, any of natural and synthetic media that contain acarbon source, a nitrogen source, an inorganic salt, or the likeutilizable by the microbes and permit efficient culture of thetransformants can be used as a culture medium. Examples of the culturemedium for E. coli include an LB medium. The transformants can beusually cultured at 37° C. for 6 to 24 hours under aerobic conditionssuch as shake culture or aeration-stirring culture. Preferably, the pHis kept around neutral pH during the culture period. If necessary, themedium may be supplemented with an antibiotic such as ampicillin ortetracycline.

When the transformants for MGAT5B expression are mammalian cells or thelike, these transformants can be cultured in a medium suitable for eachcell. The medium may or may not contain serum. A serum-free medium ismore desirable for the culture.

When the MGAT5B expression vector is, for example, a proteinexpression-inducible vector containing a repressor gene and an operator,etc., the transformants are required to induce the expression of theMGAT5B polypeptide fragment by predetermined treatment. The method forinducing the expression differs depending on a protein expressioncontrol system contained in the vector and can therefore involveinduction treatment suitable for the system. For example, the proteinexpression control system most generally used for the proteinexpression-inducible vector in bacterial hosts is a system composed of alac repressor gene and a lac operator. This system can induce expressionby IPTG (isopropyl-1-thio-β-D-galactoside) treatment. The transformantshaving the MGAT5B expression vector containing this system can expressthe MGAT5B of interest by the addition of IPTG in an appropriate amount(e.g., final concentration: 1 mM) into the medium.

(Recovery of MGAT5B Polypeptide Fragment)

Next, the MGAT5B polypeptide fragment produced in the host cells isrecovered from the cells or the culture supernatant thereof. When theproduced MGAT5B polypeptide fragment is accumulated within the microbialbodies or the cells, the microbial bodies or the cells are disrupted toextract the protein. When the MGAT5B polypeptide fragment is produced tothe outside of the microbial bodies or the outside of the cells, theculture solution may be used directly or a supernatant may be used afterremoval of the microbial bodies or the cells by centrifugation or thelike. Then, the MGAT5B polypeptide fragment can be isolated and purifiedusing a general protein purification method. The MGAT5B polypeptidefragment expressed as a fusion peptide with a labeling peptide (tag) canbe isolated and purified using, for example, affinity chromatographysuitable for each labeling peptide. Alternatively, the MGAT5Bpolypeptide fragment expressed without the labeling peptide can beisolated and purified using, for example, an ammonium sulfatesalting-out method, gel chromatography, ion-exchange chromatography,hydrophobic chromatography, or isoelectric focusing chromatography.Alternatively, two or more of these purification methods may beappropriately combined in the isolation and purification.

Finally, whether or not the MGAT5B polypeptide fragment of interest hasbeen successfully recovered can be confirmed by SDS-PAGE or the like.The recombinant MGAT5B polypeptide fragment prepared by the above methodretains glycosyltransferase activity and is soluble.

(2) Animal Immunization and Preparation of Anti-MGAT5B PolyclonalAntibody

The obtained MGAT5B polypeptide fragment can be used as an immunogen toobtain an anti-MGAT5B polyclonal antibody that specifically recognizesthe polypeptide.

First, the MGAT5B polypeptide fragment is dissolved in a buffer solutionto prepare an immunogen solution. If necessary, an adjuvant may be addedthereto for efficient immunization. For example, a Freund's completeadjuvant (FCA), a Freund's incomplete adjuvant (FIA), an aluminumhydroxide gel, a Bordetella pertussis vaccine, Titer Max Gold (Vaxel,Inc.), or GERBU adjuvant (GERBU Biotechnik GmbH) can be used alone or asa mixture as the adjuvant.

Next, a mammal is immunized by the administration of the preparedimmunogen solution. The animal used in the immunization is notparticularly limited. For example, a non-human mammal, morespecifically, a mouse, a rat, a hamster, a guinea pig, a rabbit, a goat,a donkey, sheep, a camel, a horse, or the like can be used. Hereinafter,the immunization method according to the present invention will bedescribed specifically by taking a mouse as an example.

Examples of the method for administering the immunogen solution include,but not limited to, subcutaneous injection using FIA or FCA,intraperitoneal injection using FIA, and intravenous injection usingsaline. Alternatively, the immunogen solution may be administered byintracutaneous injection or intramuscular injection. The single dose ofthe immunogen is appropriately determined according to the type of theanimal to be immunized, an administration route, etc. In the case of amouse, approximately 50 to 200 μg of the immunogen can be usuallyadministered to a 4- to 10-week-old individual. The intervals betweenimmunization shots are not particularly limited and are intervals ofseveral days to several weeks, preferably 1 to 4 weeks. After theinitial immunization, booster immunization is preferably performed. Thenumber of booster shots is 2 to 6, preferably 3 to 4. After the initialimmunization or later, blood is collected from the eye ground or thelike of the immunized mouse, and an antibody titer in the serum ispreferably measured by ELISA or the like. If a sufficient rise in theantibody titer can be confirmed, the immunogen solution can beintravenously or intraperitoneally injected to the mouse as the finalimmunization. Preferably, no adjuvant is used in the final immunization.Three to ten days, preferably 3 days, after the final immunization,blood is collected from the immunized mouse, and the serum can betreated according to a method known in the art (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988) to obtain the anti-MGAT5Bpolyclonal antibody.

(3) Preparation of Anti-MGAT5B Monoclonal Antibody

The anti-MGAT5B monoclonal antibody can be prepared according to themethod of Kohler & Milstein (Nature 256: 495-497 (1975)). For example,hybridomas are prepared by the cell fusion between antibody-producingcells obtained from the immunized animal and myeloma cells. A cloneproducing the anti-MGAT5B monoclonal antibody can be selected from theobtained hybridomas to prepare the monoclonal antibody of interest.Hereinafter, a specific example of the preparation will be described.However, the preparation of the antibody of the present invention is notlimited to the method described below.

(Collection of Antibody-producing Cell)

First, antibody-producing cells are collected from the immunized mouse.This collection is preferably performed 2 to 5 days after the finalimmunization day. Examples of the antibody-producing cells includespleen cells, lymph node cells, and peripheral blood cells. Spleen cellsor local lymph node cells are preferred. The method for collecting theantibody-producing cells from the mouse can be performed according to atechnique known in the art.

(Preparation of Hybridoma)

Subsequently, the antibody-producing cells can be fused with myelomacells to prepare hybridomas producing the anti-MGAT5B monoclonalantibody.

The myeloma cells used in the cell fusion are not particularly limitedas long as the myeloma cells are of a generally available mouse-derivedestablished cell line and can be proliferated in vitro. For convenienthybridoma screening in a step mentioned later, the myeloma cellspreferably have drug selectivity and have the property of being unableto survive in an unfused state in a selective medium and being able togrow therein only in a state fused with the antibody-producing cells.

Various cell lines already known in the art are preferably used as themyeloma cells, for example, P3 (P3×63Ag8.653) (Kearney J. F. et al.,1979, J. Immunol., 123: 1548-1550), P3×63Ag8U.1 (Yelton D. E. et al.,1978, Curr. Top. Microbiol. Immunol., 81: 1-7), NS-1 (Kohler G. et al.,1976, Eur. J. Immunol., 6: 511-519), MPC-11 (Margulies D. H. et al.,1976, Cell, 8: 405-415), SP2/0 (Shulman M. et al., 1978, Nature, 276:269-270), FO (de St. Groth S. F. et al., 1980, J. Immunol. Methods, 35:1-21), 5194 (Trowbridge I. S. 1978, J. Exp. Med., 148: 313-323), or R210(Galfre G. et al., 1979, Nature, 277: 131-133). These cell lines areavailable from RIKEN BioResource Center, ATCC (American Type CultureCollection), or ECACC (European Collection of Cell Cultures). These celllines can be cultured and subcultured according to a culture methodknown in the art (e.g., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; and Selected Methods in Cellular Immunology,W.H. Freeman and Company, 1980). Examples of the selective mediuminclude a HAT medium (RPMI1640 medium supplemented with 100 units/mLpenicillin, 100 μg/mL streptomycin, 10% fetal bovine serum (FBS), 10⁻⁴ Mhypoxanthine, 1.5×10⁻⁵ M thymidine, and 4×10⁻⁷ M aminopterin).

For the cell fusion between the myeloma cells and the antibody-producingcells, the obtained spleen cells and the myeloma cells are washed. Then,the myeloma cells and the antibody-producing cells can be mixed at amixing ratio of 1:1 to 1:10 in a medium for animal cell culture such asa MEM, DMEM, or RPMI1640 medium or a commercially available medium forcloning or cell fusion (preferably, serum-free) and contacted with eachother at 30 to 37° C. for 1 to 15 minutes in the presence of a cellfusion promoter. For example, polyethylene glycol (hereinafter, referredto as “PEG”) having an average molecular weight of 1,500 to 4,000 Da canbe used as the cell fusion promoter at a concentration of approximately10 to 80%. Alternatively, a fusion promoter or a fusion virus such aspolyvinyl alcohol or Sendai virus may be used. Usually, PEG having anaverage molecular weight of 1,500 Da is preferably used. If necessary,an aid such as dimethyl sulfoxide may be used in combination therewithin order to enhance fusion efficiency. Alternatively, theantibody-producing cells and the myeloma cells may be fused using acommercially available cell fusion apparatus based on electricstimulation (e.g., electroporation) (Nature, 1977, Vol. 266, 550-552).

After the cell fusion treatment, the cells are washed with the mediumused in the myeloma cell fusion (e.g., RPMI1640 medium). Then, a cellsuspension is prepared. Subsequently, the cell suspension isappropriately diluted with, for example, an RPMI1640 medium containingFBS and then added at a density of approximately 1×10⁴ cells/well to a96-well plate. The selective medium is added to each well where thecells can then be cultured with the selective medium appropriatelyreplaced with a fresh one. The culture temperature is 20 to 40° C.,preferably approximately 37° C. In the case of using an HGPRT-deficientor thymidine kinase (TK)-deficient line as the myeloma cells, onlyhybridomas of the antibody-producing cells and the myeloma cells can beselectively grown and proliferated by use of a selective mediumcontaining hypoxanthine, aminopterin, and thymidine (HAT medium). Thus,cells grown from approximately 10 days after the start of the culture inthe selective medium can be selected as hybridomas.

Next, the culture supernatants of the proliferated hybridomas arescreened for a hybridoma containing the anti-MGAT5B monoclonal antibodyof interest. For the hybridoma screening, for example, a part of theculture supernatant contained in the well of each cultured hybridoma canbe collected and screened by enzyme immunoassay (ELISA, etc.),radioimmunoassay (RIA), or the like with the binding activity againstthe MGAT5B polypeptide fragment used as an immunogen as an index. Theantibody-producing hybridomas are cloned in order to obtain a hybridomafurther stably producing the monoclonal antibody. The cloning can beperformed by a usual method such as a limiting dilution method or afluorescence-activated cell sorter method without particularlimitations. These screening and cloning methods can be combined tofinally establish hybridomas as anti-MGAT5B monoclonalantibody-producing cells.

If necessary, cross reactivity may be tested. Specifically, only ahybridoma producing an antibody that exhibits acceptable crossreactivity is selected by study on its binding activity against otherproteins including other glycosyltransferases, etc. The acceptable crossreactivity means the nonspecific binding activity of the monoclonalantibody at a negligible level for the use of interest.

Specific examples of the anti-MGAT5B monoclonal antibody-producinghybridoma thus selected by the screening method include GT131-2(National Deposition No: FERM P-22097; International Accession No: FERMABP-11496), GT131-7 (National Deposition No: FERM P-22098; InternationalAccession No: FERM ABP-11497), GT131-12 (National Deposition No: FERMP-22099; International Accession No: FERM ABP-11498), and GT131-18(National Deposition No: FERM P-22100; International Accession No: FERMABP-11499). These hybridomas were nationally deposited on Apr. 4, 2011with International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) and internationally deposited on thesame date with International Patent Organism Depositary, NationalInstitute of Technology and Evaluation (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan (post code: 305-8566)).

These hybridoma cell lines can be preferably cultured at 37° C. using anRPMI1640 medium supplemented with 10% FBS.

(Recovery of Anti-MGAT5B Monoclonal Antibody)

The anti-MGAT5B monoclonal antibody can be recovered by a commontechnique. The anti-MGAT5B monoclonal antibody can be recovered from theestablished hybridoma by the adoption of, for example, a usual cellculture method or an ascites formation method. The cell culture methodinvolves: culturing the anti-MGAT5B monoclonal antibody-producinghybridoma in an animal cell culture medium such as an RPMI1640 mediumcontaining 10% FBS, a MEM medium, or a serum-free medium, for example,at 37° C. for 2 to 10 days in a 5% CO₂ atmosphere; and obtaining theantibody from the culture supernatant thereof. The ascites formationmethod involves intraperitoneally administering the anti-MGAT5Bmonoclonal antibody-producing hybridoma at a dose of approximately10,000,000 cells to an animal of the same species (in the case of theabove method, a mouse) as the mammal from which the myeloma cells arederived to proliferate the hybridoma in large amounts. One to two weekslater, the ascitic fluid or serum of the animal can be collected torecover the monoclonal antibody of interest. A “GT131-2 antibody”, a“GT131-7 antibody”, a “GT131-12 antibody”, and a “GT131-18 antibody” canalso be obtained by such a method from GT131-2, GT131-7, GT131-12, andGT131-18, respectively, listed above as specific examples of theanti-MGAT5B monoclonal antibody-producing hybridoma.

The antibody can be purified, if necessary, using an appropriatepurification method known in the art. The antibody can be purifiedusing, for example, ion-exchange chromatography, affinity chromatographyusing protein A or protein G, gel chromatography, or an ammonium sulfatesalting-out method.

1-3. Epithelial Ovarian Cancer Detection Reagent

The anti-MGAT5B antibody of the present invention, a recombinantantibody for detection of an epithelial ovarian cancer marker, and/or anantibody fragment for detection of an epithelial ovarian cancer markermentioned later specifically react with the epithelial ovarian cancermarker and as such, can be used as an active ingredient in an epithelialovarian cancer detection reagent. This detection reagent can also beused to detect the epithelial ovarian cancer marker contained in asample collected from a test subject, thereby diagnosing epithelialovarian cancer developed in the test subject.

The detection reagent of the present invention can be used in any meansusing an immunological approach. The detection reagent of the presentinvention can be used in combination with, for example, a reagent for afully automatic immunoassay apparatus (e.g., chemiluminescent enzymeimmunoassay (CLEIA) apparatus) to diagnose epithelial ovarian cancerconveniently and rapidly. This will be described in detail in the fourthembodiment. Also, the epithelial ovarian cancer detection reagent can beused for staining of epithelial ovarian cancer tissues. For example, thepresence or absence of the epithelial ovarian cancer marker in tissuesobtained at laparotomy or by needlestick can be detected byimmunostaining to determine the presence or absence of epithelialovarian cancer developed in the subject. Alternatively, the presence orabsence of the epithelial ovarian cancer marker can be detected by amethod such as Western blotting using extracts of the collected tissuesto determine the presence or absence of epithelial ovarian cancerdeveloped in the subject.

1-4. Effect

The anti-MGAT5B antibody of the present invention is capable ofspecifically recognizing and binding to the intra-Golgi region of theepithelial ovarian cancer marker glycosyltransferase MGAT5B. Thus, useof the anti-MGAT5B antibody of the present invention achieves efficientdetection of the epithelial ovarian cancer marker from a sample of atest subject.

2. Recombinant Antibody for Detection of Epithelial Ovarian CancerMarker

2-1. Definition and Constitution

The second embodiment of the present invention relates to a recombinantantibody for detection of an epithelial ovarian cancer marker. Therecombinant antibody of the present invention comprises at least one setof corresponding light chain complementarity determining regions (CDRs)and heavy chain CDRs in the antibody for detection of an epithelialovarian cancer marker according to the first embodiment. In the presentspecification, the “recombinant antibody for detection of an epithelialovarian cancer marker” is also referred to as a “recombinant anti-MGAT5Bantibody”.

In the present specification, the “recombinant antibody” refers to, forexample, a chimeric antibody, a humanized antibody, and a syntheticantibody.

The “chimeric antibody” refers to an antibody derived from a certainantibody by the replacement of its light chain and heavy chain constantregions (C regions) with light chain and heavy chain C regions ofanother antibody. The chimeric antibody corresponds to, for example, anantibody derived from the mouse anti-human MGAT5B monoclonal antibodyGT131-2 antibody, GT131-7 antibody, GT131-12 antibody, or GT131-18antibody by the replacement of its C regions with C regions of anappropriate human antibody. Specifically, the chimeric antibody hasCDR-containing variable regions (V regions) derived from the GT131-2antibody, the GT131-7 antibody, the GT131-12 antibody, or the GT131-18antibody and C regions derived from the human antibody.

The “humanized antibody”, also called reshaped human antibody, refers toa mosaic antibody derived from a non-human mammal antibody, for example,an anti-human MGAT5B mouse antibody, by the replacement of only its Vregion CDRs with CDRs of an appropriate human antibody. For example, DNAsequences encoding CDR regions (CDR1 to CDR3) derived from the GT131-2antibody, the GT131-7 antibody, the GT131-12 antibody, or the GT131-18antibody are replaced with DNA sequences encoding corresponding CDRsderived from the human antibody to prepare a recombinant antibody gene,which can then be expressed to obtain a recombinant antibody that mimicsthe properties of the particular antibody. A general gene recombinationapproach for preparing the humanized antibody is also known (EuropeanPatent Application Publication No. EP 125023). Examples of suchapproaches include a method which involves: designing DNA sequences sothat mouse antibody CDRs are linked to human antibody framework regions(FRs); and synthesizing the DNA sequences by PCR using a fewoligonucleotide primers prepared to have a part overlapping with theterminal regions of both CDR— and FR-encoding sequences.

The “synthetic antibody” refers to an antibody synthesized using achemical method or a recombinant DNA method. The synthetic antibodycorresponds to, for example, a monomeric polypeptide molecule comprisingone or more light chain V regions (VLs) of a particular antibody and oneor more heavy chain V regions (VHs) thereof artificially linked via alinker peptide or the like having an appropriate length and sequence, ora multimeric polypeptide thereof. Specific examples of such polypeptidesinclude a single chain fragment of variable region (scFv) (see PierceCatalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, Ill.), adiabody, a triabody, and a tetrabody. In an immunoglobulin molecule, VLand VH are typically located on separate polypeptide chains (light chainand heavy chain). The scFv refers to a synthetic antibody fragmenthaving a structure in which the V regions on these two polypeptidechains are linked via a flexible linker having a sufficient length toform a single polypeptide chain. Both the V regions in the scFv can formone functional antigen-binding site by the self-assembly of theseregions. The scFv can be obtained by a technique known in the art whichinvolves integrating a recombinant DNA encoding the scFv into a phagegenome and expressing the DNA. The diabody refers to a molecule having astructure based on the structure of a scFv dimer (Holliger et al., 1993,Proc. Natl. Acad. Sci. USA 90: 6444-6448). For example, when the linkeris shorter than approximately 12 amino acid residues in length, twovariable domains in the scFv cannot self-assemble. By contrast, theformation of the diabody, i.e., the interaction between two scFvs, canallow VL on one Fv chain to assemble with VH on the other Fv chain toform two functional antigen-binding sites (Marvin et al., 2005, ActaPharmacol. Sin. 26: 649-658). Alternatively, cysteine residues may beadded to the C termini of scFvs to form a stable diabody through thedisulfide bond between these two Fv chains (Olafsen et al., 2004, Prot.Engr. Des. Sel. 17: 21-27). Although the diabody is such a divalentantibody fragment, its two antigen-binding sites do not have to bind tothe same epitope and may be bispecific to respectively recognize andspecifically bind to different epitopes. The triabody and the tetrabodyhave trimeric and tetrameric structures, respectively, based on the scFvstructure, as with the diabody. The triabody and the tetrabody aretrivalent and tetravalent antibody fragments, respectively, and may bemultispecific antibodies.

2-2. Preparation of Recombinant Anti-MGAT5B Antibody

The recombinant anti-MGAT5B antibody can be prepared by a DNA cloningtechnique known in the art using the anti-MGAT5B antibody prepared inthe first embodiment. For example, each literature cited above as wellas Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory ManualSecond Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. can be referred to.

2-3. Effect

The recombinant anti-MGAT5B antibody of the present invention is capableof specifically recognizing and binding to the intra-Golgi region of theepithelial ovarian cancer marker glycosyltransferase MGAT5B, as with theanti-MGAT5B antibody of the first embodiment. Thus, use of therecombinant anti-MGAT5B antibody of the present invention achievesefficient detection of the epithelial ovarian cancer marker from asample of a test subject.

3. Antibody Fragment for Detection of Epithelial Ovarian Cancer Marker

3-1. Definition and Constitution

The third embodiment of the present invention relates to an antibodyfragment for detection of an epithelial ovarian cancer marker.

The “antibody fragment” of the present invention refers to a partialregion of the anti-MGAT5B antibody according to the first embodiment orthe recombinant anti-MGAT5B antibody according the second embodiment.This partial region is a polypeptide chain having activity substantiallyequivalent to the MGAT5B-specific recognition and binding activity ofthe antibody, or a complex of the polypeptide chain. Specifically, theantibody fragment corresponds to a partial antibody region containing atleast one of the antigen-binding site(s) contained in the anti-MGAT5Bantibody of the first embodiment or the anti-MGAT5B antibody of thesecond embodiment, i.e., a polypeptide chain having at least one set ofVL and VH, or a complex thereof. Examples of the fragment of theanti-MGAT5B antibody of the first embodiment include a large number ofsufficiently characterized fragments formed by the cleavage of theantibody with various peptidases. Specific examples of the antibodyfragment include Fab, F(ab′)₂, and Fab′. The Fab refers to a fragmentthat is formed by the papain cleavage of an IgG molecule at a siteN-terminal to the hinge disulfide bond and composed of a polypeptideconsisting of VH and CH1 (VH-adjacent domain among three domains (CH1,CH2, and CH3) constituting the heavy chain constant region (H chain Cregion; hereinafter, referred to as “CH”)) and a light chain. TheF(ab′)₂ refers to a Fab′ dimer that is formed by the pepsin cleavage ofan IgG molecule at a site C-terminal to the hinge disulfide bond. TheFab′ has a structure substantially equivalent to that of Fab except thatthe H chain is slightly longer than that of Fab by containing the hingeregion. The Fab′ can be obtained by the reduction of F(ab′)₂ under mildconditions and the subsequent cleavage of the disulfide bond in thehinge region. All of these antibody fragments contain an antigen-bindingsite and are therefore able to specifically bind to the antigen (i.e.,MGAT5B in the present invention).

3-2. Effect

The antibody fragment for detection of an epithelial ovarian cancermarker of the present invention is capable of specifically recognizingand binding to the intra-Golgi region of the epithelial ovarian cancermarker glycosyltransferase MGAT5B, as with the anti-MGAT5B antibody ofthe first embodiment. Thus, use of the antibody fragment for detectionof an epithelial ovarian cancer marker of the present invention achievesefficient detection of the epithelial ovarian cancer marker from asample of a test subject.

4. Method for Diagnosis of Epithelial Ovarian Cancer

The fourth embodiment of the present invention relates to a method fordiagnosis of epithelial ovarian cancer. The method for diagnosis ofepithelial ovarian cancer according to the present invention comprisesquantitatively and/or qualitatively detecting an epithelial ovariancancer marker present in a sample derived from a test subject, anddetermining the presence or absence of epithelial ovarian cancerdeveloped in the test subject on the basis of the detection results.

4-1. Definition and Constitution

In the present specification, the “test subject” refers to an individualto be subjected to examination in the method of the present invention,i.e., an individual who provides a sample mentioned later. The testsubject is preferably an individual who may have epithelial ovariancancer or an epithelial ovarian cancer patient. In the presentspecification, the “normal individual” refers to a normal individual ina broad sense who is normal in terms of epithelial ovarian cancer, i.e.,an individual having no epithelial ovarian cancer. Thus, the normalindividual may have other diseases, for example, gastric cancer oruterine cancer as long as the normal individual has no epithelialovarian cancer. The normal individual is preferably a normal individualin a narrow sense who has no disease, i.e., a healthy individual.

The “sample” refers to a material that is collected from the testsubject and directly subjected to the method of the present invention.The sample corresponds to, for example, a body fluid, a peritoneallavage fluid, or a tissue. The “body fluid” refers to a biologicalsample in a liquid state collected directly from the test subject.Examples thereof include blood (including serum, plasma, andinterstitial fluid), lymph, spinal fluid, ascitic fluid, pleuraleffusion, sputum, lacrimal fluid, nasal discharge, saliva, urine,vaginal fluid, and semen. The “tissue” includes sections and extracts.In the method of the present invention, the sample is preferably a bodyfluid such as blood, lymph, or ascitic fluid, or a peritoneal lavagefluid.

The sample can be collected according to a method known in the art. Forexample, blood or lymph can be obtained according to a blood collectionmethod known in the art. Specifically, peripheral blood can be collectedby injection to a peripheral vein or the like. Alternatively, asciticfluid or the peritoneal lavage fluid can be collected by transabdominalultrasound-guided aspiration steering around the intestinal tract orcollected by aspiration using a syringe or the like from the Douglas'pouch after intraperitoneal injection of approximately 100 mL of salineduring abdominal section. The tissue can be collected by direct needlingto the organ or collected from a site resected during surgery. Thesample thus collected from the test subject can be used, if necessary,after dilution or concentration or after pretreatment such as theaddition of an anticoagulant such as heparin or fixation with a fixativesuch as paraffin (in the case of the tissue). Alternatively, the samplemay be used directly without such pretreatment.

The sample may be used immediately after collection or may be used bytreatment such as thawing, if necessary, after cryopreservation for agiven period. In the method of the present invention, a volume of 10 μLto 100 μL usually suffices for epithelial ovarian cancer markerdetection in the case of using serum or a peritoneal lavage fluid as thesample.

In the method of the present invention, the epithelial ovarian cancermarker to be detected is the MGAT5B polypeptide fragment described inthe first embodiment. Specifically, the epithelial ovarian cancer markeris a polypeptide fragment consisting of an amino acid sequence that islocated in an intra-Golgi region and corresponds to the C-terminalregion of MGAT5B. The epithelial ovarian cancer marker corresponds to,for example, the whole or a part of a polypeptide consisting of theamino acid sequence represented by SEQ ID NO: 1 in human MGAT5B.

4-2. Method for Detecting Epithelial Ovarian Cancer Marker

The method for detecting the epithelial ovarian cancer marker in thesample may be any method as long as the method used is known in the artand is capable of detecting the polypeptide marker. The method ispreferably a detection method based on immunological reaction(immunological detection method) using an antibody that specificallyrecognizes and binds to the epithelial ovarian cancer marker.

The anti-MGAT5B antibody according to the first embodiment (includingthe anti-MGAT5B polyclonal antibody and the anti-MGAT5B monoclonalantibody), the recombinant anti-MGAT5B antibody according to the secondembodiment, and/or the antibody fragment according to the thirdembodiment can be used as the epithelial ovarian cancer marker-specificantibody (hereinafter, referred to as an “anti-epithelial ovarian cancermarker antibody”). Specifically, examples thereof include the mouseanti-human MGAT5B monoclonal antibodies GT131-2 antibody, GT131-7antibody, GT131-12 antibody, and GT131-18 antibody described in thefirst embodiment.

Examples of the immunological detection method for detecting the amountof the epithelial ovarian cancer marker present in the testsubject-derived sample include enzyme immunoassay (including ELISA andEIA), fluorescent immunoassay, radioimmunoassay (RIA), luminescentimmunoassay, a surface plasmon resonance (SPR) method, a quartz crystalmicrobalance (QCM) method, immunoturbidimetry, latex agglutinationimmunoassay, latex turbidimetry, hemagglutination, a particleagglutination method, a gold colloid method, capillary electrophoresis,Western blotting, and an immunohistochemical method (immunostainingmethod). All of these methods are known in the art and can be performedaccording to usual methods in the art as a rule.

When the epithelial ovarian cancer marker of the present invention isassayed by the immunoassay using labeling such as enzyme immunoassay,fluorescent immunoassay, radioimmunoassay, or luminescent immunoassay,the immunological reaction is preferably performed after immobilizationof the anti-epithelial ovarian cancer marker antibody or the like orafter immobilization of a component (i.e., the epithelial ovarian cancermarker) in the sample.

An insoluble carrier having a shape such as beads, a microplate, a testtube, a stick, or a test piece made of a material such as polystyrene,polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinylchloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose,Sepharose, glass, metal, ceramics, or magnetic materials can be used asa solid-phase carrier. The immobilization can be achieved by the bindingof the anti-epithelial ovarian cancer marker antibody or the epithelialovarian cancer marker to the solid-phase carrier according to a methodknown in the art such as a physical adsorption method, a chemicalbinding method, or combined use thereof.

Examples of the label used for labeling the anti-epithelial ovariancancer marker antibody in enzyme immunoassay include peroxidase (POD),alkaline phosphatase, β-galactosidase, urease, catalase, glucoseoxidase, lactate dehydrogenase, amylase, and biotin (or avidin).Examples of the label in fluorescent immunoassay include fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, substitutedrhodamine isothiocyanate, dichlorotriazine isothiocyanate, Alexa, andAlexa Fluor. Examples of the label in radioimmunoassay include tritium,iodine 125, and iodine 131. In luminescent immunoassay, for example,NADH, FMNH2, a luciferase system, a luminol-hydrogen peroxide-PODsystem, an acridinium ester system, or a dioxetane compound system canbe used as the label.

The labeling material can be bound to the antibody by a method known inthe art such as a glutaraldehyde method, a maleimide method, a pyridyldisulfide method, or a periodic acid method for enzyme immunoassay or bya method known in the art such as a chloramine T method or aBolton-Hunter method for radioimmunoassay. The assay procedures can beperformed according to a method known in the art (Current protocols inProtein Sciences, 1995, John Wiley & Sons Inc.; and Current protocols inImmunology, 2001, John Wiley & Sons Inc.).

The immunoassay includes a direct detection method which involveslabeling the anti-epithelial ovarian cancer marker antibody andquantifying the epithelial ovarian cancer marker in the sample by directdetection, and an indirect detection method which involves quantifyingthe epithelial ovarian cancer marker in the sample by indirect detectionusing a labeled secondary antibody. Either of the methods may be used inthe present invention.

In the case of the direct detection method, for example, the polypeptidefragment in the sample is immobilized on the carrier and contacted withthe labeled anti-epithelial ovarian cancer marker antibody to form acomplex of the epithelial ovarian cancer marker of the present inventionand the labeled anti-epithelial ovarian cancer marker antibody. Then,unbound labeled antibodies are washed off. From the amount of the boundlabeled antibody or the amount of the unbound labeled antibody, theepithelial ovarian cancer marker in the sample can be detected and theamount thereof can be measured.

In the case of the indirect detection method, the epithelial ovariancancer marker in the sample is reacted with the anti-epithelial ovariancancer marker antibody as a primary antibody (primary reaction) andfurther reacted with a labeled secondary antibody (secondary reaction).The primary reaction and the secondary reaction may be performed inreverse order or may be performed simultaneously. The labeled secondaryantibody may specifically recognize and bind to the primary antibody ormay recognize and bind to the epithelial ovarian cancer marker. Theprimary reaction and the secondary reaction form a complex consisting ofthe immobilized epithelial ovarian cancer marker, the anti-epithelialovarian cancer marker antibody, and the labeled secondary antibody or acomplex consisting of the immobilized anti-epithelial ovarian cancermarker antibody, the epithelial ovarian cancer marker, and the labeledsecondary antibody.

The indirect detection method involving forming the complex of theimmobilized anti-epithelial ovarian cancer marker antibody, theepithelial ovarian cancer marker, and the labeled secondary antibody iscalled sandwich method. This method detects the antigen of interestusing two different types of antibodies capable of concurrently bindingto the same antigen, i.e., two or more types of monoclonal antibodies(or at least one type of polyclonal antibody) that recognize differentepitopes on one antigen. So-called sandwich ELISA, which involvesimmobilizing one of these two types of antibodies onto the carrier,labeling the other antibody, and detecting the antigen of interest, isfrequently used as the sandwich method. This method can also bepreferably used in the method of the present invention. After thecomplex formation, proteins other than the epithelial ovarian cancermarker contained in the sample and unbound labeled secondary antibodiesare washed off. From the amount of the bound labeled secondary antibodyor the amount of the unbound labeled secondary antibody, the epithelialovarian cancer marker in the sample can be detected and the amountthereof can be measured. The indirect detection method based on thesandwich method is preferably used because the epithelial ovarian cancermarker in the sample can be assayed highly precisely without sacrificingsensitivity. This approach is also applicable to automatization using anexisting automatic immunological detection apparatus.

A preferred exemplary embodiment of the detection of the epithelialovarian cancer marker of the present invention will be shown. First, theanti-epithelial ovarian cancer marker antibody of the present invention(e.g., any one of the GT131-2 antibody, the GT131-7 antibody, theGT131-12 antibody, and the GT131-18 antibody) is immobilized as aprimary antibody onto the insoluble carrier. After the immobilization,the antibody-nonimmobilized solid-phase surface is blocked with aprotein irrelevant to the antigen epithelial ovarian cancer marker(e.g., fetal calf serum, bovine serum albumin, and gelatin) or anon-protein blocking agent such as sucrose or a chemically synthesizedpolymer. Next, the immobilized primary antibody is contacted with thetest subject-derived sample. Subsequently, another anti-epithelialovarian cancer marker antibody (in the above case, any one antibody(other than the antibody selected above) selected from the groupconsisting of the GT131-2 antibody, the GT131-7 antibody, the GT131-12antibody, and the GT131-18 antibody) that recognizes and binds to anepitope different from that for the primary antibody on the epithelialovarian cancer marker is contacted therewith as a secondary antibody.The secondary antibody used is a labeled secondary antibody obtained bythe above labeling method. Then, unbound labeled secondary antibodiesare removed by washing. Then, a signal derived from the label on thelabeled secondary antibody bound with the carrier can be detected.

4-3. Diagnosis of Developed Epithelial Ovarian Cancer

Epithelial ovarian cancer developed in the test subject is determined onthe basis of detection results obtained by quantitatively and/orqualitatively detecting the epithelial ovarian cancer marker present inthe test subject-derived sample.

Examples of the qualitative detection of the epithelial ovarian cancermarker include detection by an immunohistochemical method or Westernblotting.

In the case of quantitatively detecting the epithelial ovarian cancermarker, for example, by immunoassay, epithelial ovarian cancer isdiagnosed on the basis of the quantitative assay results. When themeasurement value of the MGAT5B polypeptide fragment is equal to orhigher than a predetermined value, the test subject is diagnosed to belikely to have epithelial ovarian cancer.

In the present specification, the “measurement value” refers to a valueobtained by the above assay method and may be an absolute value such asconcentration or may be a relative value such as signal intensity fromthe epithelial ovarian cancer marker per unit sample.

Usually, the glycosyltransferase MGAT5B polypeptide fragment found as anepithelial ovarian cancer marker by the present inventors is rarelydetected in a sample derived from a normal individual. Thus, the testsubject can be diagnosed to be likely to have epithelial ovarian canceras a rule provided that the MGAT5B polypeptide fragment of interest isdetected.

If more highly precise diagnosis is desired, a method based on themeasurement value is preferred. Specifically, when the MGAT5Bpolypeptide fragment is detected by the assay method with a measurementvalue equal to or higher than a predetermined value, the test subjectcan be diagnosed to be likely to have epithelial ovarian cancer. The“predetermined value” refers to a measurement value capable ofseparating epithelial ovarian cancer patients from normal individuals,for example, a cutoff value. Specifically, examples of the predeterminedvalue in the case of using the anti-epithelial ovarian cancer markerantibody of the present invention include a cutoff value mentionedlater.

The anti-epithelial ovarian cancer marker antibody used, however, mayhave cross reactivity, albeit slightly, and detect otherglycosyltransferases. Alternatively, nonspecific reaction derived fromimpurities may not be eliminated for normal individuals or patients withcancer other than epithelial ovarian cancer. In such cases, only thedetection of the presence or absence of the epithelial ovarian cancermarker might not achieve accurate diagnosis.

Thus, for example, if the anti-epithelial ovarian cancer marker antibodyused may have cross reactivity, the epithelial ovarian cancer marker canbe detected by the sandwich method to thereby enhance its specificityand also significantly reduce the nonspecific reaction derived fromimpurities. The GT131-2 antibody, the GT131-7 antibody, the GT131-12antibody, and the GT131-18 antibody of the present invention have allbeen confirmed to exhibit no cross reactivity.

If there is the possibility that the epithelial ovarian cancer marker ofthe present invention is detected even in a normal individual, thepresence or absence of epithelial ovarian cancer may be diagnosed on thebasis of a statistically significantly high measurement value of theepithelial ovarian cancer marker in the test subject-derived samplecompared with that in the normal individual. The value of the epithelialovarian cancer marker obtained by the above quantitative method can beused as the measurement value of the epithelial ovarian cancer marker.In this case, a protein known in the art expected to have noquantitative difference between the samples of the test subject and thenormal individual can be used as an internal control to correct thequantitative detection results of the test subject and the normalindividual. Thus, the amount of the epithelial ovarian cancer marker canbe obtained more accurately. Examples of such internal control proteinsinclude albumin.

The term “statistically significantly” means that the test subject andthe normal individual exhibit significant difference therebetween whenthe quantitative difference in the epithelial ovarian cancer markercontained in their samples is statistically treated. Specifically,examples of the significant difference include difference with asignificance level smaller than 5%, 1%, or 0.1%. A test method known inthe art capable of determining the presence or absence of significancecan be appropriately used as a testing method for the statisticaltreatment without particular limitations. For example, the student's ttest or multiple comparison test method can be used. The term“statistically significantly high” specifically means that the obtainedvalue is higher than a cutoff value defined as a value capable ofseparating individuals having epithelial ovarian cancer from normalindividuals, etc. such that sensitivity and specificity set by a routinemethod in multiple-specimen analysis are optimized.

In the case of using, for example, the GT131-7 antibody and the GT131-12antibody as the anti-epithelial ovarian cancer marker antibodies in thesandwich method according to the present invention, the cutoff value atwhich individuals having epithelial ovarian cancer are separated fromnormal individuals is 4.0 ng/mL (the upper 95 percentile of theperitoneal lavage fluids of disseminated gastric cancer patients),preferably 4.6 ng/mL (the optimal threshold point in the drawn ROC curveof the peritoneal lavage fluids of ovarian cancer patients vs. theperitoneal lavage fluids of disseminated gastric cancer patients), morepreferably 14.3 ng/mL (the lower 95 percentile of the peritoneal lavagefluids of ovarian cancer patients). Thus, when the measurement value ofthe epithelial ovarian cancer marker in the test subject-derived sampleis higher than the cutoff value, the test subject can be diagnosed to belikely to have epithelial ovarian cancer.

EXAMPLES Example 1 Preparation of Anti-MGAT5B Antibody and HybridomaProducing the Antibody

1. Selection and Preparation of Immunogen

An immunogen MGAT5B gene was selected by real-time PCR analysis ontranscripts of a total of 186 glycosyltransferases known in the art inovarian cancer cell lines RMG-I, RMG-II, and RMG-V (all derived fromclear cell adenocarcinoma), RMUG-S (derived from mucinousadenocarcinoma), peripheral blood cells, and normal tissues of the largeintestine, the liver, and the stomach. Specifically, their transcriptionlevels in the ovarian cancer cell lines and the normal tissues wereranked by the comparison of mean values and the comparison of maximumvalues to select a top glycosyltransferase as an ovarian cancer markercandidate. MGAT5B, one of the selected candidates, was ranked in firstin terms of both the mean value and the maximum value and had a muchhigher transcription level in ovarian cancer cells than in normaltissues. In addition, MGAT5B was placed in a higher rank than that ofGAT (mean value: No. 14 and maximum value: No. 13) andβ1,3-galactosyltransferase 4 (mean value: No. 32 and maximum value: No.17) and therefore selected as a promising marker candidate specific forovarian cancer.

For the expression of the MGAT5B gene, a MGAT5B expression vector wasprepared on the basis of pIRESpuro3 (Clontech Laboratories, Inc.).First, pFC3 (Promega K.K.) was cleaved with EcoRI and EcoRV. Next, aplasmid containing a full-length human MGAT5B gene sequence (SEQ ID NO:5) was obtained by a method described in Non Patent Literature (KanekoM. et al., 2003, FEBS Letters, 554: 515-519). This plasmid was used as atemplate in PCR amplification using primer A (aaagaattccGGGGACTCGC: SEQID NO: 6) and primer B (TCACAGACAGCCCTG: SEQ ID NO: 7). This PCR wasperformed by 30 cycles each involving 94° C. for 30 seconds, then 55° C.for 30 seconds, and 68° C. for 30 seconds. The amplification product waspurified using Qiagen Minielute Kit, then digested with EcoRI and EcoRV,and inserted to the cleaved pFC3 (pFC3-MGAT5B). The obtained plasmidpFC3-MGAT5B encodes a fusion protein (soluble MGAT5B polypeptidefragment) composed of: a region from Gly 51 to 372 Leu in MGAT5Brepresented by SEQ ID NO: 1; and preprotrypsin (MSALLILALVGAAVA: SEQ IDNO: 3), FLAG (DYKDDDDK: SEQ ID NO: 4), and LAAANSE (linker) linked tothe N-terminus thereof. The preprotrypsin sequence is cleaved off duringsecretion from cells.

Subsequently, pFC3-MGAT5B was cleaved with restriction enzymes NruI andBamHI and inserted to similarly cleaved pIRESpuro3 (ClontechLaboratories, Inc.). In this way, an expression vectorpIRES-F-puro3-MGAT5B was obtained.

pIRES-F-puro3-MGAT5B was transferred to E. coli DH5α and subclonedtherein, followed by transfection of HEK293T by a routine method usingLipofectamine 2000. A stably expressing strain was obtained by selectionin the presence of 10 μg/mL puromycin. The soluble MGAT5B polypeptidefragment of interest was recovered using Anti-FLAG M2 Affinity Gel(A2220; Sigma-Aldrich Corp.). Its expression was then detected bySDS-PAGE and confirmed by Western blotting using Monoclonal Anti-FLAG M2Antibody Affinity purified (F1804; Sigma-Aldrich Corp.).

For the large-scale preparation of the soluble MGAT5B polypeptidefragment, the strain was cultured at 37° C. in 1500 mL of a medium (DMEMhigh glucose, 10% FBS, 10 μg/mL puromycin, 500 U/mL penicillin, 10 μg/mLstreptomycin). The culture was started at 15% confluence and continuedfor approximately 2 days. Then, the culture supernatant was recovered ina 70% confluent state.

Insoluble residues were removed from the obtained culture supernatantusing Nalgene PES Bottle Top Filter, 0.45 μm cut-off. 1.5 mL ofAnti-FLAG M2 Affinity Gel (Sigma-Aldrich Corp.) was added into thesample, and the mixture was left with stirring at 4° C. The resin wasrecovered, then packed into an empty column, and washed with 25 mL ofPBS, followed by elution using 1 mL of a FLAG peptide (manufactured bySigma-Aldrich Corp.) dissolved at a concentration of 1 g/mL in PBS. Theeluted fraction was concentrated into 500 μL by ultrafiltration andsubsequently applied to a Superdex 200 10/300 GL (GE Healthcare JapanCorp.) gel filtration column equilibrated in advance with a buffersolution (20 mM Tris-HCl, 50 mM NaCl, pH 8.0). 250 μL each of fractionswas separated, and each fraction was electrophoresed to obtain afraction containing the soluble MGAT5B polypeptide fragment. Thesefractions were combined and then concentrated into 2.5 mg/mL with thesolvent replaced with PBS to obtain a soluble MGAT5B polypeptidefragment preparation. This preparation was used as an immunogen.

2. Immunization and Cell Fusion

50 μg of the soluble MGAT5B polypeptide fragment thus prepared as animmunogen was dissolved in 0.1 mL of saline and then supplemented andfully mixed with 0.1 mL of a Freund's complete adjuvant to prepare anemulsion. 0.2 mL of the emulsion was subcutaneously injected to the backof a Balb/c mouse (9-week-old female). 0.05 mL of an aluminum adjuvantwas added to an immunogen solution containing 25 μg of the immunogendissolved in 0.05 mL of saline, and fully mixed, and the resultingemulsion was intraperitoneally injected to the mouse 1 week after theinitial immunization. A solution containing 50 μg of the immunogendissolved in 0.1 mL of saline and a solution containing 25 μg of theimmunogen dissolved in 0.05 mL of saline were further intraperitoneallyinjected to the mouse 4 weeks after the initial immunization and 5 weeksafter the initial immunization, respectively.

Three days after the final immunization, the spleen was excised from themouse, and extracted spleen cells were washed with an RPMI1640 medium. Asuspension of 1.2×10⁸ spleen cells and a suspension of 3×10⁷ mousemyeloma cells (P3/X63-Ag8.U1) were mixed and centrifuged, followed byremoval of the medium. 2 mL of polyethylene glycol/RPMI1640 mediumheated to 37° C. was gradually added to the cells, which were then fusedby mild stirring. Then, the medium was removed by centrifugation, and20% S-Clone/Cloning Medium (Sanko Junyaku Co., Ltd.) and 60 mL of anRPMI1640 medium containing 10% FBS were added to the cells.Subsequently, the mixture was dispensed at a volume of 0.1 mL/well to a96-well plate. Four hours later, a HAT medium (HAT medium×1concentration: 0.1 mM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine,20% S-Clone, RPMI1640 medium containing 10% FBS) adjusted to twice theusual concentration was added thereto at a volume of 0.1 mL/well. Themedium in each well was replaced by half with a fresh one 2 days and 4days after the fusion. After 10-day culture, hybridoma growth wasobserved in approximately 80% of the wells.

3. Selection of Hybridoma

For screening for an antibody in each hybridoma culture supernatant,study was made using ELISA with the MGAT5B polypeptide fragment preparedin the paragraph “1. Selection and preparation of immunogen” as anantigen. First, the antigen was adsorbed at a concentration of 1 μg/mLin PBS onto a microtiter plate for ELISA. The plate was blocked with PBScontaining 5% sucrose and 5% Tween 20 and then reacted with thehybridoma culture supernatant. The plate was further reacted with aperoxidase-labeled goat anti-mouse immunoglobulin antibody. Theabsorbance was measured at 450 nm using 3,3′,5,5′-tetramethylbenzidine(TMB) as a substrate to detect the antibody of interest. As a result,antibody-producing hybridomas were obtained in a total of 18 wells.These hybridomas were each transferred to an HT medium (HAT mediumexcept for aminopterin), further transferred to an RPMI1640 mediumcontaining 10% FBS, and cultured.

Next, the hybridomas were cloned by the limiting dilution method. Eachhybridoma was diluted into a density of approximately 1 cell/well in a96-well plate and cultured in an RPMI1640 medium containing 15% FBS.Approximately 10 days later, the growth and number of colonies in thewells were confirmed by microscopic examination. Approximately 14 dayslater, 0.1 mL of each culture supernatant was recovered and used toselect antibody-producing cells by ELISA.

Also, reactivity with impurities that might be contained in theimmunogen was confirmed by ELISA. FBS for culture used in the paragraph“1. Selection and preparation of immunogen” and an arbitrary polypeptidefragment (different from the immunogen) expressed using the same vectoras in the FLAG-MGAT5B polypeptide fragment were each adsorbed at aconcentration of 2 μg/mL in PBS onto a microtiter plate for ELISA.Hybridomas whose culture supernatants reacted therewith were discarded.In this way, 22 stable monoclonal antibody-producing hybridomas wereobtained as a result of cloning.

These 22 hybridomas thus obtained were further cultured. Immunoglobulinadsorbed onto a protein-G agarose gel (GE Healthcare Japan Corp.) waspartially purified from 10 mL of each culture supernatant. TheFLAG-MGAT5B polypeptide fragment, a MGAT5B polypeptide fragment fromwhich the FLAG tag was cleaved off by enzymatic treatment, an arbitrarypolypeptide fragment different therefrom expressed using the same vectoras in the FLAG-MGAT5B polypeptide fragment, and FBS were eachelectrophoresed under SDS-PAGE reduced conditions and transferred to aPVDF membrane. Each prepared membrane was reacted with thehybridoma-derived immunoglobulin adjusted to a concentration of 1 μg/mLby Western blotting. Eleven hybridomas exhibited strong reactivity inthe Western blotting. Four hybridomas exhibited weak reactivity in theWestern blotting, but strong reactivity in ELISA. The immunoglobulinsubclasses of these monoclonal antibodies strongly reacted in Westernblotting or ELISA were determined using a mouse monoclonal antibodyisotyping kit (AbD Serotec, A Bio-Rad Company). All of these antibodieswere IgG1, κ.

Of the anti-MGAT5B monoclonal antibody-producing hybridomas thusobtained, 6 hybridomas (GT131-2, GT131-7, GT131-9, GT131-12, GT131-14,and GT131-18) having a feature such as particularly strong reactivity inWestern blotting or ELISA were further used to prepare antibodies inlarge amounts.

Specifically, each hybridoma was expanded into 100 mL in an RPMI1640medium containing 10% FBS and recovered at the logarithmic growth phase.The cells were dispersed in 2% FBS and 500 mL of an RPMI1640 mediumcontaining ITS-A (10 mg/L insulin, 6.7 mg/L sodium selenate, 5.5 mg/Ltransferrin, 11.0 mg/L sodium pyruvate) and rotary cultured in a rollerbottle. At day 2 and day 4 after the start of the rotary culture, thecells were further diluted twice with an ITS-A-containing RPMI1640medium and maintained until day 10 to day 14. Finally, a culturesupernatant derived from 1% FBS and 1 L of the ITS-A-containing RPMI1640medium was obtained in two roller bottles. The cells were removed bycentrifugation and filtration. Then, NaCl, glycine, sodium azide, andsodium hydroxide were added to the supernatant to adjust pH at 8.9containing 3 M NaCl, 1.5 M glycine, and 0.1% sodium azide. Then, theculture supernatant was applied to a protein A-Sepharose affinitycolumn, 4 mL (GE Healthcare Japan Corp.) equilibrated with the samebuffer solution as above (containing 3 M NaCl, 1.5 M glycine, and 0.1%sodium azide, pH 8.9) to bind the antibody to the column. Next, thecolumn was washed with the above buffer solution in an amount of 20times the column, followed by elution with a 0.1 M citrate buffersolution (pH 6.0) containing 0.1% sodium azide. Peaks were fractionatedusing a fraction collector. Each peak fraction thus obtained wasrecovered and then salted out with 50% saturated ammonium sulfate. Aftercentrifugation, the precipitate was dissolved in a 50 mM tris buffersolution (pH 8.0) containing 0.15 M NaCl and 0.1% sodium azide. In thisway, approximately 10 to 40 mg of purified IgG was obtained from each ofthe 6 hybridomas.

The hybridomas GT131-2, GT131-7, GT131-12, and GT131-18 were depositedwith International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, as mentioned above.

Example 2 Confirmation of Antigen Specificity of Monoclonal Antibody

The hybridoma-derived anti-MGAT5B monoclonal antibodies (GT131-12antibody and GT131-18 antibody) obtained in Example 1 were studied fortheir antigen specificity.

(Method)

10 ng of the FLAG-MGAT5B polypeptide fragment obtained in Example 1, 10ng of the MGAT5B polypeptide fragment obtained by the cleavage of theFLAG tag from the fragment through enzymatic treatment, 1 μL ofimmunoglobulin- and albumin-free serum from a pool normal human serum(NHS) containing a serum mixture from a plurality of healthy adults, and0.5 μL of the pool NHS were each electrophoresed using a 10%polyacrylamide gel under SDS-PAGE reduced conditions and transferred toa PVDF membrane. Each membrane was blocked with PBS containing 5%skimmed milk and then reacted with each purified MGAT5B monoclonalantibody (1 μg/mL) at room temperature for 90 minutes. After washing,the membrane was soaked with an HRP-labeled anti-mouse IgG antibody (GEHealthcare Japan Corp.) as a secondary antibody and reacted at roomtemperature for 60 minutes. After washing, the antigen-antibody bindingwas detected on the basis of chemiluminescence using a Western blottingdetection reagent (PerkinElmer Inc.).

(Results)

The results are shown in FIG. 1. The GT131-12 antibody and the GT131-18antibody were both confirmed to sufficiently bind to the FLAG-MGAT5Bpolypeptide fragment and the MGAT5B polypeptide fragment from which theFLAG tag was cleaved off. By contrast, these antibodies exhibited weak(GT131-12 antibody) or no (GT131-18 antibody) reactivity with the NHS.These results demonstrated that the GT131-12 antibody and the GT131-18antibody specifically react with the MGAT5B polypeptide fragment and donot react with other proteins contained in human serum.

Example 3 Detection of MGAT5B Polypeptide Fragment byImmunoprecipitation Using Anti-MGAT5B Antibody

(Method)

The pool NHS was diluted 100-fold with a 50 mM tris buffer solution (pH8.0) containing 0.15 M NaCl and 0.1% sodium azide. The FLAG-MGAT5Bpolypeptide fragment prepared in Example 1 was added thereto at a finalconcentration of 10 μg/mL. This mixture was used as animmunoprecipitation sample. 2 μg of each monoclonal antibody (GT131-2antibody, GT131-7 antibody, GT131-12 antibody, or GT131-18 antibody) and20 μL of a protein G gel (GE Healthcare Japan Corp.) were added to 0.5mL of this sample and mixed at 4° C. for 4 hours using a rotator. Thesample solution was discarded by centrifugation, and the protein G gelwas washed with the above tris buffer solution. Then, 40 μL of anSDS-PAGE sample buffer was added thereto, and the mixture was heated at98° C. for 5 minutes to obtain immunoprecipitated fractions.

Subsequently, the FLAG-MGAT5B polypeptide fragment, each fractionimmunoprecipitated with the GT131-2 antibody, the GT131-7 antibody, theGT131-12 antibody, or the GT131-18 antibody, and a mixture of anon-immunoprecipitated pool NHS and the FLAG-MGAT5B polypeptide fragmentwere each electrophoresed using a 10% polyacrylamide gel under SDS-PAGEreduced conditions and transferred to a PVDF membrane. Each membrane wasblocked with PBS containing 5% skimmed milk and then reacted with anHRP-labeled anti-FLAG antibody (M2, Sigma-Aldrich Corp.) at roomtemperature for 1 hour. Alternatively, the membrane was reacted with theGT131-12 antibody (2 μg/ml) or the GT131-18 antibody (1 μg/ml) labeledusing a biotin labeling kit (Dojindo Laboratories) at room temperaturefor 2 hours. After washing, the membrane was reacted with HRP-labeledstreptavidin (GE Healthcare Japan Corp.) at room temperature for 60minutes. These PVDF membranes were washed and then subjected todetection based on chemiluminescence using a Western blotting detectionreagent (PerkinElmer Inc.).

(Results)

The results are shown in FIG. 2. The FLAG-MGAT5B polypeptide fragmentadded as a sample was detected from all of the immunoprecipitatedfractions. These results showed that the monoclonal antibody of thepresent invention, i.e., the GT131-2 antibody, the GT131-7 antibody, theGT131-12 antibody, or the GT131-18 antibody is capable of specificallyimmunoprecipitating the MGAT5B polypeptide fragment even from a proteinsolution rich in impurities, such as serum.

Example 4 Immunostaining of Ovarian Cancer Cell Line Using Anti-MGAT5BAntibody

An ovarian cancer cell line was immunostained using the anti-MGAT5Bmonoclonal antibodies (GT131-2 antibody and GT131-18 antibody) preparedin Example 1.

(Method)

The culture supernatant (containing the GT131-2 antibody) of the GT131-2antibody-producing hybridoma, the culture supernatant (containing theGT131-18 antibody) of the GT131-18 antibody-producing hybridoma, or ananti-B4GALT1 monoclonal antibody MAb8628 (Uemura M. et al., 1992, CancerRes., 52: 6153-6157) for use as a trans-Golgi marker was added to anepithelial ovarian cancer cell line RMUG-S and reacted overnight at 4°C. Then, the binding reaction of the antibodies with variousglycosyltransferases was visualized using Alexa 488-labeled anti-mouseIgG (Life Technologies Corp.). The nuclei were stained with Hoechst33342 (Life Technologies Corp.). Images were taken using a Keyencemicroscope BioZero.

(Results)

The results are shown in FIG. 3. FIG. 3a is a staining pattern of theepithelial ovarian cancer cell line RMUG-S immunostained with theGT131-2 antibody. FIG. 3b is a staining pattern of the cell lineimmunostained with the GT131-18 antibody. FIG. 3c is a staining patternof the cell line immunostained with the staining positive controlanti-B4GALT1 antibody. The antibody-stained part is indicated by anarrow, and the nuclear-stained part is indicated by an arrowhead. Fromthese results, the GT131-2 antibody and the GT131-18 antibody were bothconfirmed to be capable of staining the epithelial ovarian cancer cellsin a manner specific for the epithelial ovarian cancer marker. Theintracellular localization pattern of the glycosyltransferase detectedwith the anti-B4GALT1 antibody was the same as that of theglycosyltransferase detected with the GT131-2 antibody or the GT131-18antibody. This suggests that MGAT5B is a glycosyltransferase localizedto the Golgi bodies. These results showed that the GT131-2 antibody andthe GT131-18 antibody of the present invention can be used in thestaining of epithelial ovarian cancer cells and tissues.

Example 5 Selection of Antibody for Sandwich ELISA Assay System

(Method)

Six anti-MGAT5B antibodies (GT131-2 antibody, GT131-7 antibody, GT131-9antibody, GT131-12 antibody, GT131-14 antibody, and GT131-18 antibody)obtained in Example 1 were each used as an antibody for immobilizationon an ELISA plate and as an antibody for detection to study a sandwichELISA assay system. First, each antibody was diluted into 4 μg/mL withPBS and added at a volume of 100 μL/well to a microplate for ELISA. Eachantibody was adsorbed onto the plate overnight at 4° C. Then, thesolution was discarded, and each well was washed. Next, PBS containing3% bovine serum albumin (BSA) was added thereto as a blocking solutionat a volume of 300 μL/well to block the plate. The blocking solution wasdiscarded, and each well was washed. Then, 100 μL of a MGAT5Bpolypeptide fragment solution adjusted to 0, 31.25, 62.5, 125, 250, or500 ng/mL was added to each well. After reaction at 37° C. for 2 hours,the solution in each well was discarded, and each well was washed. Then,six antibodies (GT131-2 antibody, GT131-7 antibody, GT131-9 antibody,GT131-12 antibody, GT131-14 antibody, and GT131-18 antibody)biotinylated using a biotin labeling kit (Dojindo Laboratories) wereeach adjusted to 1 μg/mL and reacted therewith at room temperature for 2hours. Then, the solution was discarded, and each well was washed. Then,a horseradish peroxidase (HRP)-labeled avidin (The Jackson Laboratory)solution was added thereto at a volume of 100 μL/well and reacted atroom temperature for 1 hour. The reaction solution was discarded, andeach well was washed. Then, color developed by a TMB substrate solution(Pierce Chemical Co.) was measured on the basis of absorbance at 450 nm.One (GT131-9) of these 6 monoclonal antibodies obtained in Example 1 wasweak reactive. In addition, two antibodies (GT131-14 and GT131-18) wereunreactive in sandwich ELISA using each other and were thereforepresumed to recognize the same site. The other 4 monoclonal antibodiesGT131-2, GT131-7, GT131-12, and GT131-18 exhibited MGAT5Bconcentration-dependent reactivity in any combination, showing thatthese 4 antibodies can be used in a sandwich ELISA assay system. Theresults are shown in FIG. 4. Among them, the monoclonal antibodyGT131-12 for immobilization on a microplate and the monoclonal antibodyGT131-7 for detection were used in the subsequent Examples as apreferred combination for detecting the antigen MGAT5B.

Example 6 Construction of Highly Sensitive Sandwich CLEIA System

In order to enhance the sensitivity of the sandwich ELISA system usingthe combination of the monoclonal antibodies selected in Example 5,i.e., the GT131-7 antibody and the GT131-12 antibody, the application ofa chemiluminescent detection system (chemiluminescent enzymeimmunoassay; CLEIA) was studied.

(Method)

The GT131-12 antibody of the present invention was adjusted to 4 μg/mL,then added at a volume of 100 μL/well to a 96-well microplate forfluorescent photometry (Nunc, Thermo Fisher Scientific Inc.), andimmobilized thereon at room temperature for 7 hours. After discarding ofthe antibody solution, the plate was washed with PBS containing 0.05%Tween 20 and blocked overnight at 4° C. by the addition of a blockingsolution (20 mM tris (pH 8.0) containing 0.2% highly pure casein(I-Block, Life Technologies Corp.), 0.1% Tween 20, and 0.15 M NaCl) at avolume of 300 μL/well. The MGAT5B polypeptide fragment was adjusted as aconcentration standard to 0, 0.6125, 1.25, 2.5, 5, 10, 20, 40, 80, and160 ng/mL with the above blocking solution. 10 μL of each concentrationstandard was added, together with 90 μl, of the blocking solution, toeach well of the microplate thus washed, and reacted at 37° C. for 2hours. The plate thus reacted was washed five times with PBS containing0.05% Tween 20. The GT131-7 antibody labeled with alkaline phosphataseusing an alkaline phosphatase labeling kit (Dojindo Laboratories) wasadjusted to 0.5 μg/mL with the above blocking solution, then added tothe plate at a volume of 100 μL/well, and reacted at room temperaturefor 1.5 hours in the dark. The plate thus reacted was washed four timeswith PBS containing 0.05% Tween 20 and twice with 20 mM tris (pH 9.8)containing 1 mM magnesium chloride. A chemiluminescence reagent CSPDSubstrate Sapphire-II (Life Technologies Corp.) was added thereto at avolume of 100 μL/well and reacted at room temperature in the dark. 45minutes later, luminescence intensity was measured.

(Results)

FIG. 5 shows a calibration curve obtained from the measurement values ofthe concentration standard. Sandwich CLEIA using the GT131-12 antibodyand the GT131-7 antibody of the present invention was confirmed to becapable of quantifying the glycosyltransferase MGAT5B polypeptidefragment in a test subject-derived sample with detection sensitivity ofapproximately 1 ng/mL. The concentration of the MGAT5B polypeptidefragment present in a sample obtained from a test subject can bedetermined using this calibration curve.

Example 7 Measurement of Amount of MGAT5B Polypeptide Fragment inPeritoneal Lavage Fluid of Epithelial Ovarian Cancer Patient, Etc.

(Method)

(1) Experimental Example 1

40 peritoneal lavage fluids collected from epithelial ovarian cancerpatients as well as 55 peritoneal lavage fluids collected from patientswith intraperitoneally disseminated gastric cancer and 159 peritoneallavage fluids of patients with non-intraperitoneally-disseminatedgastric cancer as control groups were used as samples in sandwich CLEIAusing the calibration curve of the MGAT5B polypeptide fragment obtainedin Example 6. The concentration of the epithelial ovarian cancer marker(MGAT5B polypeptide fragment) in each sample was quantitativelymeasured. The detailed assay method was performed according to themethod described in Example 6.

(2) Comparative Example 1

40 peritoneal lavage fluids collected from the epithelial ovarian cancerpatients used in Experimental Example 1 were used as samples to assayexisting ovarian cancer markers CA125 and GAT in each sample. CA125 andGAT were assayed using Abnova CA125 (Human) ELISA kit (catalog No:KA0205) and Konica Minolta GAT Test Kit, respectively, according to theprotocols attached thereto. The respective obtained measurement valuesof the markers were compared with the concentration measurement value ofthe MGAT5B polypeptide fragment in 40 peritoneal lavage fluids of theovarian cancer patients obtained in Experimental Example 1.

(Results)

The results of Experimental Example 1 are shown in FIG. 6. The resultsof Comparative Example 1 are shown in FIGS. 7 and 8.

First, referring to FIG. 6, the measurement values (epithelial ovariancancer marker concentrations) were statistically analyzed andconsequently significantly different among the above 3 groups.Particularly, the ovarian cancer group exhibited significantly highmeasurement values compared with two gastric cancer groups. The meanvalue thereof was 19.6 ng/mL in the specimens of the ovarian cancerpatients, 3.3 ng/mL in the specimens of the disseminated gastric cancerpatients, and 2.1 ng/mL in the specimens of the non-disseminated gastriccancer patients. These results demonstrated that sandwich CLEIA usingthe two anti-MGAT5B antibodies of the present invention, i.e., theGT131-7 antibody and the GT131-12 antibody, with a cutoff value set to4.6 ng/mL can determine epithelial ovarian cancer developed in the testsubject from the sample derived therefrom with high accuracy rate ofdiagnosis.

Next, referring to FIG. 7, the concentration of the MGAT5B polypeptidefragment exhibited correlation with neither of the measurement values ofthe existing ovarian cancer markers CA125 (a) and GAT (b). By contrast,as shown in FIG. 8, positive correlation was observed between the CA125measurement value and the GAT measurement value with a correlationcoefficient of 0.65. These results suggested that the measurement valueof the MGAT5B polypeptide fragment according to the present inventionreflects a disease marker secreted under a mechanism different from thatof the existing ovarian cancer markers.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

DEPOSITION NUMBER

GT131-2; National Deposition No: FERM P-22097; International AccessionNo: FERM ABP-11496

GT131-7; National Deposition No: FERM P-22098; International AccessionNo: FERM ABP-11497

GT131-12; National Deposition No: FERM P-22099; International AccessionNo: FERM ABP-11498

GT131-18; National Deposition No: FERM P-22100; International AccessionNo: FERM ABP-11499

The invention claimed is:
 1. A method for diagnosis of epithelialovarian cancer, comprising: (a) quantitatively detecting aβ-1,6-N-acetylglucosaminyltransferase 5B polypeptide fragment present ina sample derived from a test subject using at least oneanti-β-1,6-N-acetylglucosaminyltransferase 5B monoclonal antibody, orantibody fragment thereof, and (b) determining that the test subject isdiagnosed to be likely to have epithelial ovarian cancer when theresults of quantitatively detecting theβ-1,6-N-acetylglucosaminyltransferase 5B polypeptide fragment aregreater than a predetermined cut-off value, wherein the predeterminedcut-off value is the 95th percentile of the quantitative detectionresults of the β-1,6-N-acetylglucosaminyltransferase 5B polypeptidefragment in the sample derived from normal individuals or patients otherthan epithelial ovarian cancer patients, wherein theanti-β-1,6-N-acetylglucosaminyltransferase 5B monoclonal antibody isproduced by a hybridoma identified by International Accession No. FERMBP-11496, FERM BP-11497, FERM BP-11498, or FERM BP-11499.
 2. The methodfor diagnosis of epithelial ovarian cancer of claim 1, wherein theβ-1,6-N-acetylglucosaminyltransferase 5B polypeptide fragment comprisingan epitope recognized by the anti-β-1,6-N-acetylglucosaminyltransferase5B monoclonal antibody, or the antibody fragment thereof is the whole ora part of a polypeptide consisting of the amino acid sequence of SEQ IDNO:
 1. 3. The method for diagnosis of epithelial ovarian cancer of claim1, wherein two anti-β-1,6-N-acetylglucosaminyltransferase 5B monoclonalantibodies are used to detect the β-1,6-N-acetylglucosaminyltransferase5B polypeptide fragment.
 4. A method for diagnosis of epithelial ovariancancer, comprising: (a) quantitatively detecting aβ-1,6-N-acetylglucosaminyltransferase 5B polypeptide fragment present ina sample derived from a test subject using at least one monoclonalantibody or antibody fragment thereof, and (b) determining that the testsubject is diagnosed to be likely to have epithelial ovarian cancer whenthe results of quantitatively detecting theβ-1,6-N-acetylglucosaminyltransferase 5B polypeptide fragment aregreater than a predetermined cut-off value of 4.0 ng/mL, wherein themonoclonal antibody is produced by a hybridoma identified byInternational Accession No. FERM BP-11496, FERM BP-11497, FERM BP-11498,or FERM BP-11499.
 5. The method for diagnosis of epithelial ovariancancer of claim 1, wherein the sample is a body fluid, a peritoneallavage fluid, or a tissue.
 6. The method for diagnosis of epithelialovarian cancer of claim 4, wherein twoanti-β-1,6-N-acetylglucosaminyltransferase 5B monoclonal antibodies areused to detect the β-1,6-N-acetylglucosaminyltransferase 5B polypeptidefragment.